                1. HIV and AIDS Epidemic        
Infection with Human Immunodeficiency Virus (HIV), a pathogenic retrovirus, can cause Acquired Immunodeficiency Syndrome (AIDS) (Barre-Sinossi, F. et al., 1983, Science 220: 868-870). Although macrophage, neuron and other cells can be infected by HIV (Maddon et al., 1986, Cell 47:333-48), the CD4+ lymphocytes are the major target cells for HIV (Dalgleish, A. et al., 1984, Nature 312:767-8), because HIV has strong affinity to the CD4 molecules on the surfaces of CD4+ cells. HIV infection in a human body destroys so many CD4+ lymphocytes that the body begins to lose its immune function, therefore an AIDS patient is highly vulnerable to various infections, neuronal dysfunction, tumors, and so on. Suffering from the symptoms, the patients die eventually (edited by Levy, J. A.: Acute HIV infection and susceptible cells, published in U.S.A, 2000, Page 63-78).
With its severe symptoms and high mortality rate, the epidemic contagion of AIDS has become one of the leading causes of death that is threatening human health. So far in the entire world, people infected by HIV have accumulated to a total of 57,900,000. 21,800,000 people have died from AIDS in the last decade. 5,300,000 people were found to have newly contracted HIV within the year 2000. In China, HIV infection spreads rapidly. Experts estimated that in 2000 the population of HIV positives has exceeded 800,000-1,000,000, which includes both adults and children (WHO Report 2000, UNAIDS and WHO).
Currently at least two types of HIV have been identified: HIV-1 (Gallo, R. et. al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 223:343-346). Each of them has high genetic heterogeneity. For HIV-1 alone, there are at least 11 different genotype (A-J and O subtypes) (Jonassen, T. O. et al., 1997, Virol. 231:43-47). The E subtype of HIV-1 is distributed mainly in Central Africa, Thailand, India, Vietnam, Kampuchea, Malaysia, Burma, China, and western hemisphere (WHO Report 1996). The HIV subtypes found in China are mostly B, E, or C subtype (Yu, E. S. et al., 1996, American J. Public Health 86(8 Pt1): 1116-22).
The reproduction cycle of HIV has several important steps. First, the envelope glycoprotein gp120 attaches itself to the host cell membrane through its specific binding with CD4 molecule located on the surface of T4 lymphocyte. With the assistance of chemokine co-receptor, the viral envelope fuses with the host cellular membrane (Berger, E. A., et al., 1999, Ann. Rev. Immunol., 17: 657-700). After the fusion process, the HIV virion packed in nucleocapsid enters into the host cell with its capsid shucked off and viral nucleic acid exposed. The viral reverse transcriptase catalyzed the transcription of HIV single-stranded RNA into single-stranded DNA, which is then transformed to double-stranded DNA by the catalysis of cellular polymerase. The double-stranded DNA can either exist freely in cytoplasm or be integrated as provirus into host chromosome DNA by the catalysis of viral integrase, thus engendering HIV latent infection (Roe, T. et al., 1997, J. Virol. 71(2):1334-40). Provirus, which will not be excised from the host chromosome, is very stable and reproduces itself with the replication of host chromosome. After the HIV mRNA is translated into a large polyprotein, the viral proteases cut and process the polyprotein to form mature viral structural proteins (Xiang, Y. & Leis, J., 1997, J. Virol. 71(3): 2083-91). These structural proteins, together with HIV nucleic acids, are finally assembled into new virus granules and released outside the cell by budding (Kiss-Lazozlo, Hohn, T., 1996, Trends in Microbiology 4(12):480-5).
In summary, the critical stages of HIV replication are: 1) attachment and entry into host cell through a fusion process; 2) reverse transcription and integration; 3) protein translation and processing; 4) virus assembly and release.
2. The Treatment for HIV Infection
Although great efforts have been dedicated to effective remedial and preventive methods for many years, there is no working vaccine or cure for AIDS yet.
An ideal vaccine should be innocuous and capable of inducing neutralizing antibodies as well as persistent immune responses in mucous membrane and blood (Levy, J. A. and Levy, J. A., 1988, Trens Med. Rev. 2:265-71). Many HIV vaccines currently developed in the world are still in the stages of animal trials. Although vaccines against HIV membrane proteins gp160 and gp120 have already moved into first, second, or third stages of clinical trials, the results of the trials are disappointing. Many vaccines that are effective to prevent HIV infection in laboratory animals are not effective in human (McElrath, M. J. et al., 1996, Pro. Natl. Acad. Sci. USA 93:3972-77). The fact that scientists are making little progress in HIV vaccine research could be attributed to the complexity and variability of HIV genetic materials (Bloom, B. R., 1996, Science 272:1888-1900).
The drugs against AIDS approved in the world could be classified into two categories: HIV reverse transcriptase inhibitors (Charles, C. J., et al., 1996, JAMA 276:146) and HIV protease inhibitors (Miles, S. A. et al., International AIDS Society USA 4(3):15). Both of them aim at later stages of HIV infection—transcription and assembly of new viruses. The well-known “Cocktail Therapy” is a combination therapy using both types of inhibitors (Lafeuillade, A., et al., 1997, J. Infect. Dis. 175:1051-55).
Reverse transcriptase inhibitors, including AZT, ddI, ddC, 3TC, and d4T, etc, would induce drug resistance, sooner or later, that means the viruses become less sensitive to the drugs, and the effective inhibition concentration of the drugs rise by several-fold or even ten-fold (Vella, S. and Floridia, M., 1996, International AIDS Society USA 4 (3):15). This drug-resistance is associated with high mutation rate of HIV. In a human body, a single HIV virus could produce 108-1010 new virus granules every day, while the mutation rate is 3×105 per replication cycle. Many mis-sense mutations, affecting the expression of amino acids, may happen in the regulatory genes as well as in the envelope proteins. In some HIV strains, the mutation rate could be as high as 40% in the amino acid sequences of certain genes(Myers, G and Montaner, J. G., 1992, The Retroviridae vol. 1, Plenum Press, New York 51-105). As a result, reverse transcriptase inhibitors lead to drug-resistance by facilitating the proliferation of resistant strains that exist before and after the mutations in addition to control sensitive virus strains.
Moreover, all the reverse transcriptase inhibitors have specific toxicity related to their dosage. The symptoms include spinal cord suppression, vomiting, liver dysfunction, muscle weakness, diseases of peripheral nervous system, and pancreatic inflammation. Many patients have to suspend the treatment due to these intolerable side effects (Fischl, M. A., et al., 1987, N. Engl. J. Med. 317:185-91; Lenderking, W. R., et al., 1994, N. Engl. J. Med. 330:738-43).
Drug-resistance is also a major problem for protease inhibitors. Mutations in viral protease gene have caused drug-resistance in all the protease inhibitors presently used in AIDS treatment (Condra, J. H. et al., 1995, Nature 374:569-71). The side effects of protease inhibitors include liver dysfunction, gastrointestinal discomfort, kidney stone, numbness around mouth, abnormality of lipid metabolism, and mental disorder (Deeks, et al., 1997, JAMA 277:145-53).
In summary, most of the currently used anti-HIV drugs are highly toxic, and induce drug-resistance. Therefore, there is still a huge obstacle in the treatment of HIV infection. Apparently, it is urgent for the need of new drugs with better efficacy and lower toxicity for the treatment of HIV infection.
New drugs can be developed against new targets in different stages of HIV replication cycle. Recently a few of anti-AIDS drugs with new mechanisms have been developed after in-depth research in HIV and AIDS. These drugs include some new HIV reverse transcriptase inhibitors and HIV protease inhibitors, as well as some new anti-HIV agents aimed at other targets that are listed here (De, C. E., 2000, Rev. Med. Virol. 10 (4):255-77):
1). Virus absorbents, such as sodium lauryl sulfate, dextrose sulfate, and heparin, can interrupt the cohesion of gp120 on HIV envelope and the lymphocyte through the action of polyanion groups. However, these absorbents have bad specificity and high toxicity. Some of them can even increase the virus load (Baba, M., et al., 1988, Pro. Natl. Acad. Sci. USA. 85:6132-6);
2). Soluble CD4s are used to prevent gp120 from binding to host cells. Some recombinant soluble CD4s could bind the virus granules before gp120 contact the CD4 molecules on cellular membrane and prevent HIV infection. However, these recombinant soluble CD4s are of no apparent effect on the HIV-1 strains isolated from some patients. Moreover, the clinical experiments did not provide any reliable evidence for their antiviral activity (Gomatos, P. J. et al., 1990, J. Immunol. 144:4183-8);
3). Chemokines and their analogs, including RANTES, MIP-1α, MIP-1β binding with CCR5 and SDF binding with CXCR4, can be used to prevent HIV from entering into host cells. They could not only competitively block the gomphosis between HIV gp120 and cellular chemokine co-receptors but also limit HIV inbreak points by depressing the expression of this co-receptor on cell. The latest chemokine co-receptor blockers include positive charged small peptides such as ALX40-4C and T22, and compounds such as AMD3100, TAK-779 and trichosanthin.
4). Although soluble CD4-IgG can suppress HIV replication in vitro, it has no reliable antiviral activity in clinical trials.
5). Agents such as 2,2′-dithiobisbenzamides (DIBAs) and azadicarbonamide (ADA) can block the assembly and disassembly of viruses through interactions with NCp7 zinc finger site.
6). A segment of gp41 or its analog can be used as a fusion inhibitor. For example, T-20 is capable of blocking virus entry into the cell (Jiang, S. et al., 1993, Nature 365:113.)
7). Inhibitors of viral mRNA transcriptase, such as CGP64222, fluoroquinolone K-12, and EM2487;
8. Inhibitors of integrase, such as derivatives of Carbonyl J [N,N′-bis(2-(5-hydroxy-7-naphthalenesulfonic acid)urea], can prevent HIV from integrating its genome into host lymphocyte genome (Maurer K, et al:, 2000, Bioorg Chem 28(3):140-155).
3. Fusion Inhibitors Blocking Viral Entry Into Cells
Many biological processes involve membrane fusion. In eukaryotic cells, the fusions of cellular membranes are happening continuously, including endocytosis, secretion, recycle of membranous components, and so on (White, J. M., 1992, Science 258:917-24). Examples of fusion in some peculiar cells include the secretion of regulated fusion hormone, enzyme, and nerve transmitter. Some more notable examples include the fusion of germ cells and of muscle cells.
According to an embodiment of the present invention, the anti-fusion or anti-membrane fusion drug is an agent that inhibits or suppresses the fusion of two or more biological membranes. According to an embodiment of the present invention, two or more biological membranes are either cellular or viral structures, such as cellular membrane and viral envelope. According to an embodiment of the present invention, the antiviral agent is a compound that inhibits viral infection of cells, such as the inhibition of virus-cell fusion, or cell-cell fusion. According to an embodiment of the present invention, the infection is related to membrane fusion, such as envelope viral infection of cells, and other processes similar to viral and cellular fusion, such as what happens during bacterial conjugation.
In conclusion, membrane fusion is a critical step for envelope virus to attack and penetrate the host cells (Weissenhorn, W., et al., 1997, Nature 387:426-30). The anti-HIV drug of the present invention, Fusonex, as well as its derivatives, is a fusion blocker to prevent viruses from entering host cells.
The fusion process is controlled by the glycoproteins on HIV envelope. The precursor of the glycoproteins is gp160 that has polysaccharide groups. During the virus reproduction period, gp160 is hydrolyzed by certain protease into two subunits: gp120, which is outside the envelope, and gp41, which is a trans-membrane protein. After the hydrolyzation, gp120 and gp41 are still linked by non-covalent bonds and polymerized as trimers outside the virus granule. The trans-membrane protein gp41, whose ectodomain with a highly helical structure, has a highly efficient origination mechanism for membrane fusion, and is known as the pivotal molecule to open the gate of cells for its direct participation in the fusing process of cellular membranes (Ferrer, M., et al., 1999, Nat. Struct. Biol. 6(10):953-60; Zhou, G., et al., 2000, 1: Bioorg. Med. Chem. 8(9):2219-27).
It has been demonstrated by crystal diffraction analysis that when fusion takes place between viruses and cells, the core of gp41 is composed of six helical bundles wherein the N-terminal and C-terminal helices are collocated as three hairpins which fix the HIV envelope to the cellular membrane. While the gp41 trimer can form a fusion pore that facilitates the viral intrusion into the host cell (Chan, D. C., et al., 1997, Cell 89:263-73), it exists in an unstable natural non-fusion conformation on the surface of the free virus granule fresh sprouting from infected cells. At first, the N-terminal helix is wrapped inside the C-terminal helix so that the N-terminal fusion area is hidden, then after gp120 on viral surface combines with the CD4 receptor and chemokine co-receptor on the cellular membrane, an receptor-activated conformational change of gp41 occurs in which its N-terminal extends beyond the viral surface into the host cellular membrane. At this time, gp41 is transformed from an unstable natural non-fusion conformation into a pro-hairpin intermediate conformation. When the C-terminal and N-peptides of gp41 bind together, the hydrophobic N-terminal core of the trimer structure is exposed, and the pro-hairpin intermediate is transformed into a more energy-stabilized hairpin conformation, and by this time the viral envelope has fused with the cellular membrane (Jones, P. L., et al., 1998, J. Biol. Chem. 273:404).
The first fusion inhibitor ever discovered is a 36 amino-acid peptide derived from the C-terminal (127-162) of gp41-T-20, its sequence is as follows:
X-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-Z.
The structural similarity of T-20 to the C-terminal of gp41 makes it capable of competing with the C-peptide of gp41 in binding with its N-terminal fusion area. On the surface of T-cell, T-20 at very low concentration can interrupt the fusion between HIV gp41 and host cellular membrane (the IC50 is within the range of nM) (Jiang, S., et al., 1993, Nature 365:113; Wild, C. T. et al., 1994, Pro. Natl. Acad. Sci. USA 91:9770-74). In the pro-hairpin state which lasts many minutes, T-20 is very effective in inhibiting the binding of the C-peptide of gp41 with its N-terminal fusion area, thus blocking the formation of a hairpin between the viral envelope and cellular membrane (Kliger, Y and Shai, Y., 2000, J. Mol. Biol. 295:163-8).
Because a fusion inhibitor acts on the cellular membrane, it doesn't need to be released inside cells to exert its function. In comparison, the anti-HIV drugs, currently in clinical use, all act in the middle or late stages of viral infection of host cells, that means they must be first released into the cells to be able to inhibit the reproduction of the invading HIV. In addition, the highly conserved amino acid sequence of the hydrophobic core of gp41 suggests that the virus is not likely to develop drug resistance against the fusion inhibitors. It is shown in vitro experiments that T-20 can specifically block HIV entry into cells. On the other hand, both the first and the second stages of clinical trials have indicated that AIDS patients can put up well with T-20 administration. T-20 has no toxicity against the spinal cord, and the most side effects are in low grade, or in middle grade. In a daily dosage of 200 mg, T-20 can remarkably reduce the HIV loads in most patients, and 30% has fallen below a detectable level (lower than 400/ml). Besides, T-20 is also effective to HIV patients who have already developed drug resistance. It is reported that the number of CD4+ cells in some patients has undergone some increase after the use of T-20 (Kilby, J. M. et al., 1998, Nat. Med. 4:1302-1307). It has been worried that long term administration of T-20 might induce the production of specific antibodies against T-20, that could cause the AIDS patients resistance to T-20. Nevertheless, during an experiment of a few weeks, T-20 maintained antiviral activity all the time (Pilcher, C. D. et al., 1999, AIDS 13(15):2171-4).
Compared with the HIV reverse transcriptase inhibitors and protease inhibitors currently in clinical use, the advantages of the fusion inhibitor T-20 are better efficacy, lower toxicity, and no drug resistance yet. However, the clinical dosage of T-20 is as high as 200 mg per day is an indication of its bad stability and low anti-fusion valence. In addition, because of such high dosage, T-20 has caused some local responses in some patients (Kilby, J. M. et al., 1998, Nat. Med 4:1302-1307).